Malignant tumors accompanied with malignant pleural effusion (MPE) often indicate poor prognosis. The therapeutic effect and mechanism of intrapleural injection of anti-programmed cell death protein 1 (PD1) on MPE need to be explored.via flow cytometry, RNA-sequencing, quantitative PCR, western blot, immunohistochemistry, and other experimental methods.A preclinical MPE mouse model and a small clinical study were used to evaluate the effect of intrapleural injection of anti-PD1 antibody. The role of immune cells was observed Intrathoracic injection of anti-PD1 monoclonal antibody (mAb) has significantly prolonged the survival time of mice (P = 0.0098) and reduced the amount of effusion (P = 0.003) and the number of cancer nodules (P = 0.0043). Local CD8+ T cells participated in intrapleural administration of anti-PD1 mAb. The proportion of CD69+, IFN-γ+, and granzyme B+ CD8+ T cells in the pleural cavity was increased, and the expression of TNF-α and IL-1β in MPE also developed significantly after injection. Local injection promoted activation of the CCL20/CCR6 pathway in the tumor microenvironment and further elevated the expression of several molecules related to lymphocyte activation. Clinically, the control rate of intrathoracic injection of sintilimab (a human anti-PD1 mAb) for 10 weeks in NSCLC patients with MPE was 66.7%. Local injection improved the activity and function of patients’ local cytotoxic T cells (CTLs).Intrapleural injection of anti-PD1 mAb could control malignant pleural effusion and the growth of cancer, which may be achieved by enhancing local CTL activity and cytotoxicity. Malignant pleural effusion (MPE) refers to pleural effusion caused by the metastasis of primary pleural tumor or other malignant tumor to the pleura . ApproxiHowever, the medical talc powder that can be injected into the chest cavity has not been produced and sold in China; it is only available as a product for external use. Clinically, to reduce the recurrence of pleural effusion, the current view is that systemic treatment combined with local intervention in the pleural cavity  is also The environment of MPE is highly similar to that of the tumor microenvironment . The for. Mice were reportedly administered with an intrathoracic injection of 200μg anti-PD1 mAb (InVivoMab anti-mouse PD-1 (CD279), Bioxcell)/0.9% normal saline on the 7th and 14th day of the model. We then injected 50 μL anti-CD8 mAb  intraperitoneally multiple times to achieve CD8 depletion. The fixed mouse tissues were successively scanned using a CT machine (Siemens Somatom Sensation 16).Mouse Lewis lung carcinoma cell line (LLC) was purchased from Shanghai Cell Bank of Chinese Academy of Sciences. The MPE mouse model  has been previously described , 20 . All patients were assessed by CT and then underwent thoracic puncture drainage . IBI308  was then diluted in normal saline and injected after adequate drainage. Patients were required to return to the hospital for chest CT review at 5 and 10 weeks after discharge for assessment. NCI-CTC AE version 4.03 standard was used to evaluate the safety of drugs. Pleural fluid was collected from all patients before and after treatment.2+ collagenase (Sigma–Aldrich). Lymphocytes were isolated from the patient’s pleural effusion using blood density gradient centrifugation. The following antibodies were used: fixable viability dye eFluor 780, Alexa Fluor®488 anti-mouse CD3 antibody, mouse CD3E PERCP mAb 0.025 mg 145-2C11, PE/Cy7 anti-mouse CD45 antibody, FITC anti-mouse CD8b (Ly-3) antibody, APC anti-mouse CD69 antibody, Alexa Fluor®647 anti-mouse CD4, PE anti-mouse IFN-γ antibody, APC anti-human/mouse granzyme B recombinant antibody, APC-Cy™7 mouse anti-human CD3, FITC mouse anti-human CD8, FITC mouse anti-human CD4, PerCP mouse anti-human CD69, and PE mouse anti-human CD279.Cancerous nodules in the thoracic cavity of mice were collected after dissection and digested with the addition of Ca® UltraTM RNA Library Prep Kit was used to construct the library. Gene Ontology (GO) enrichment analysis and Reactome database analysis were performed. qPCR (Takara SYBR Premix Ex TaqTM) experiment was performed on a qPCR instrument (Quantagene q225). Primer sequences are shown in RNA extraction from tumor tissues in the mouse pleural cavity was performed using TRIzol™ Reagent (Invitrogen). RNA purity (NanoPhotometer) and concentration (Qubit 2.0 Fluorometer) were determined, and RNA integrity was then assessed . The Illumina The levels of TNF-α and IL-1β  in pleural effusions and the blood of mice were determined. Anti-CD8 antibody (Abcam), anti-PD1 antibody , anti-PDL1 antibody , anti-GAPDH antibody , and anti-β-actin antibody  were used for antigen detection. Lymphocyte and tumor cell lines were co-cultured in 96-well plates at a ratio of 20:1. The LDH release experiment was then performed using the lactate dehydrogenase cytotoxicity test kit (Beyotime Biotechnology).After collecting the clinicopathological data of the patients, statistical classification was summarized by using Excel. Flow cytometry results were re-aggregated and analyzed using FlowJo. SPSS and GraphPad prism 5.0 were used for statistical plotting. All experiments were performed with ≥ 3 biological replicates. The independent sample t-test was used to compare data between two groups. *P < 0.05, **P < 0.01, ***P < 0.001 were considered to be of significant difference, whereas ns showed no significant difference.. In addition, the average volume of pleural effusion in the anti-PD1 mAb group was less than 300 μL and that in the NS group was more than 500μL (P=0.003) . The size and number of cancerous nodules in the treatment group were determined to be lower than those in the NS group (P = 0.0043) . Although both groups experienced dramatic weight loss at the later stage of the model, no significant difference was detected . The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Cre) in the blood of mice did not vary significantly , suggesting that no obvious toxic or side effects were observed when intrapleural injection of anti-PD1 mAb was administered.The median survival of MPE model mice treated with intrapleural injection of anti-PD1 mAb was approximately 1.345-fold compared to that of the control mice treated with 0.9% saline  . In addi. In addi. In addi. In addi; it also limited the proliferation of cancer nodules to the greatest extent (P = 0.0003). However, even one-tenth (50μg) of the administered dose could also limit the amount of MPE and the number of cancerous nodules . The weight of mice in the high-dose group (500μg) was observed to decrease gradually from the second week , considering the obvious side effects. Log-rank analysis showed that each dose group had an extended survival period (P = 0.0178) . Mice treated with 200μg anti-PD1 mAb achieved the longest median survival period.Intrapleural injection of 500μg high-dose anti-PD1 mAb was the most effective dose for MPE (P = 0.0008) ; it also; it also; it also. Systemic administration was determined to have a greater impact on AST than intracavitary administration (P=0.0069) . Surprisingly, contralateral intrapleural administration  could also reduce the amount of MPE produced in mice  and the number of cancerous nodules .Interestingly, intrathoracic and caudal intravenous injections yielded similar results  . The CD8 depletion group obtained the lowest number of CD8+cells. There was no fluctuation of local CD8 infiltration even after anti-PD1 treatment . The mice in the anti-PD1 group had the least amount of MPE. However, despite simultaneous clearance of CD8 and immunotherapy, there was no improvement in controlling excessive MPE . The number of cancer nodules remained significant (P = 0.0013) after CD8 depletion, and subsequent anti-PD1 treatment did not resolve the substantial number of cancerous nodules in the chest (P = 0.0187) .CD8 depletion in mice was achieved by continuous intraperitoneal injection of anti-CD8 antibody , resulti, resulti, resulti, resulti. Intrapleural injection with anti-PD1 after CD8 depletion did not prevent the weakened IFN-γ secretion by CTLs (P = 0.0064) . The activity of CD8+T cells at the tumor site was most enhanced in the anti-PD1 mAb treatment group, via intrapleural injection, compared with those of other groups (P = 0.0337). Using flow cytometry, we observed that the proportion of CD8+ T cells releasing granzyme B peaked in the tumors of mice receiving anti-PD1 treatment . In the spleen, after CD8 depletion, IFN-γ and granzyme B secreted by CD8+ T cells were not much . In the pleural effusion of mice receiving intrapleural injection, the levels of TNF-α (P = 0.0004) and IL-1β (P = 0.0422) were significantly higher than those of the NS group . However, the concentration of these factors in the peripheral blood of mice did not differ before or after treatment . Local anti-PD1 treatment failed to change the levels of systemic inflammatory factors in mice.CD69  and intr. Intrapl. Intrapl. Intrapl. Intrapl. Intrapl. After local anti-PD1 treatment, a large number of genes including human latent-transforming growth factor beta-binding protein 2 (Ltbp2) and lymphocyte antigen 6 complex (Ly6D), and chemokine ligand 20 (Ccl20) were rapidly upregulated; while the expression of the programmed-death receptor gene Pdcd1(P = 0.031), granzyme F (Gzmf), Hba-a1, and Clec10a, etc. were all significantly down-regulated .In total, 420 genes were upregulated and 189 downregulated  ; additionally, the level of Ccr6 mRNA was also significantly increased (P = 0.0074) . Following CD8 clearance, expression of Ccl20 mRNA was slightly lower than that of the treatment group; however, expression of Ccl20 increased with both CD8 clearance and local anti-PD1 treatment. The expression of Ccr6 in the treatment group was highest (P = 0.0373), whereas this did not increase in the CD8-cleared group .In the GO and Reactome enrichment analysis of upregulated differential genes, the genes annotated to the chemotaxis pathway were the most numerous with the greatest significant difference in expression levels ture DC) . The mRN. CCL20 i. CCL20 iTcf7 (P = 0.0092), Lef1 (P = 0.0007), and Btla (P = 0.0002) mRNA expression increased more than 4 times. Expression of Cxcr5 in the control group was only 34.4% compared to that of the treatment group (P = 0.028) . There was significant inhibition of expression of Tcf7, Lef1, and Btla mRNA following CD8 depletion, and anti-PD1 therapy has increased this effect .CXCR5 , TCF7, L= 0.028) . There w. PD-L1 expression was also suppressed to a certain extent after receiving anti-PD1 treatment . We verified via western blots that PD-1 and PD-L1 protein expressions in the treatment group were significantly inhibited in adjacent tissues  and tumor tissues  in comparison with those in the control.Interestingly, with IHC staining of cancerous nodules on the pleura of different groups of mice, PD-1 staining in the low-dose group (P < 0.0001), the medium-dose group (P = 0.0419), and the high-dose group (P = 0.0021) was not as clear as that in the NS group . PD-L1 e. PD-L1 e. PD-L1 e. After adequate drainage, we injected human anti-PD1 (sintilimab) into the pleural cavity, and its effectiveness and safety in treating MPE were then evaluated . After 5 weeks of intrapleural administration, pleural effusion volume decreased substantially in seven out of nine patients. Following a further 10 weeks of intrapleural administration, the control rate of pleural effusion was still 66.7% . Adverse reactions were rare in these patients, with grade III adverse reactions occurring in only a few cases . After treatment, consistent with the results of animal experiments, there was a significant increase in the levels of CD69 (P = 0.0391), a molecule closely related to CD8+ T lymphocyte activation , and IFN-γ, which is secreted by CTLs, (P = 0.0252) . The proportion of CD8+ T cells positive for molecular granzyme B also has significantly increased following intrapleural injection of anti-PD1 mAb (P = 0.0187) . The expression of PD-1 on the surface of CTL in MPE showed a decreasing trend . The activity and killing capacity of CD8+ T cells in peripheral blood were noted to increase, but lacked significant difference .The clinicopathological characteristics of the nine advanced NSCLC patients with MPE included in this study are summarized in the table below . After a. After a. After a. After a. After a. After a. After a. After aDelaying the recurrence of malignant pleural effusion and improving survival have always been problems that need to be overcome, and there is no uniform standard in the industry. For the first time, our team attempted to improve MPE by intrathoracic injection of immune checkpoint inhibitors. Here, the median survival time of the mice in the treatment group was 10 days longer than that in the control group. Intrapleural injection of anti-PD1 mAb was demonstrated to have a therapeutic effect on MPE in mice.Intraperitoneal injection of a localized low-dose anti-CTLA-4 mAb has controlled the growth of colorectal cancer in mice and was not inferior to the administration of a systemic high-dose treatment . Our stuWe first observed extensive infiltration of CD8+ T cells in the tumor area after intrapleural injection. After CD8 removal, the effect of anti-PD1 treatment group was decreased, which further explained the critical role of CD8+ T cells in treating MPE with anti-PD1 mAb. Due to the activation effect of CD8+ T cells, CTLs are required to produce a lasting and effective antitumor immune response in MPE . We founWe have also examined the effect of treatment on two pro-inflammatory factors, TNF-α and IL-1β. The use of combined immunotherapy, including anti-PD1 therapy, can enhance the release of TNF-α and other factors from T cells to induce stronger and more effective antitumor effects . In our MPE, like the immune microenvironment of other malignancies, has a complex interplay of cells and molecules . The rolLef1 and Tcf7, which encode the transcription factors LEF-1   and follrs LEF-1  and TCF-rs LEF-1 , would trs LEF-1 . The lev T cells . The expBoth IHC and western blot data show that the expression of PD-1 in intrapleural cancer and adjacent cancer areas was significantly reduced, which may be due to the activation of PD-1 insufficient expression induction . A signiSintilimab (a human anti-PD1 mAb) has been approved for second-line systemic treatment in lymphoma by the National Medical Products Administration of China, and it has demonstrated advantages in treating NSCLC [ORIENT-11 ]. The coAt present, our hypothesis of local administration and experimental design are still relatively new attempts in the clinical tumor field of immunotherapy for MPE. We comprehensively considered this hypothesis first in a single-arm exploratory study, and the efficacy of the 9 patients included in the study was acceptable. Of course, we will also try to carry out a larger sample size multi-center study in this direction under the condition of ethical approval, and design a randomized controlled test to verify this hypothesis.From the point of view of economic cost, it is well known that the most commonly used pleurodesis is talc, the price is very cheap. However, medical talc, which can be injected into the chest, is not yet produced and sold in China. Immunotherapy is indeed more expensive than many drugs used for intraperural injection, such as cisplatin, IL-2 and endostatin. However, based on the gradual promotion and application of anti-PD1 therapy in various tumor treatments, with the gradual optimization of relevant drug production technology and the support of the government, we believe that it will become an affordable and cost-effective drug for more people in the near future.Our study is the first to propose intrapleural injection of immune checkpoint inhibitors for treating MPE, and it was experimentally validated in animal models and clinical patients. Moreover, we preliminarily revealed the complex local tumor microenvironment of MPE. To expand on these results, the application of other immune checkpoint inhibitors in pleural diseases will be explored in future work.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA761009.The original contributions presented in the study are publicly available. This data can be found here: The studies involving human participants were reviewed and approved by The Ethical Committee and Institutional Review Board of the Jinling Hospital, affiliated to Nanjing University School of Medicine . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Ethical Committee and Institutional Review Board of the Jinling Hospital, affiliated to Nanjing University School of Medicine . Written informed consent was obtained from the owners for the participation of their animals in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XL and YS conceived and designed the study. XL conducted most of the experiments. GW, CC, YZ, and SZ performed parts of the experiments. SZ, XS, and JY provided reagents and acquired data. XL, CC, and YZ analyzed data. XL and GW wrote the manuscript. TL and YS supervised the project and contributed to writing the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China (grant number 81772500) and the Key Research and Development Program of Jiangsu Province (grant number BE2019719).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Central nervous system demyelination is the main feature of multiple sclerosis (MS). The most important unmet need in MS is use of treatments that delay the progression of the disease. Leucine-rich repeat and Immunoglobulin-like domain containing NOGO receptor-interacting protein 1(LINGO-1) have been known as inhibitors of oligodendrocyte differentiation and myelination.We investigated LINGO-1 antibody effects on remyelination and neurobehavioral deficit using cuprizone-induced demyelination. Animals were randomly divided into three groups (n = 10): (1) Control group; received the regular diet, (2) CPZ group; normal saline was injected intraperitoneally, and (3) Treatment group; LINGO-1 antibody (10 mg/kg) was injected IP once every six days for 3 weeks. We assessed the level of myelin basic protein (MBP), neurofilament heavy chain (NF200), and Brain-derived neuroprotective factor (BDNF) in the corpus callosum (CC) by immunostaining against MBP, NF200, and BDNF. We found decreased levels of MBP, NF200, and BDNF in demyelinated CC, and anti-LINGO-1 treatment improved demyelinated structures. Furthermore, motor impairment was measured by Open-field (OFT) and Balance beam tests. In the treatment group, motor impairment was significantly improved. These results provide evidence that LINGO-1 antibody can improve remyelination and neurobehavioral deficit.  Multiple Sclerosis (MS) is a debilitating , demyeliThe cause of MS is unknown and pathological . There iin vitro  maturation, including pathways involving Notch-1, Wnt, LINGO-1, hyaluronan, and Retinoid X receptor (RXR). LINGO-1 inhibits maturation of oligodendrocytes , 17. Accin vitro . Clearlyin vitro . in vitro . Animals were kept under standard conditions  and had free access to water and food. All experiment procedures were performed according to the standards established by Tehran University of Medical Sciences ethical committee (approved ID: IR.TUMS.MEDICINE.REC.1399.130). Thirty male adult C57BL/6 mice weighing 20–30 g (8 weeks) were purchased from LINGO-1 treatment The LINGO-1 antibody  began in the third week, because significant demyelination is detectable after 3 weeks in the cuprizone-fed mice . Based oExperimental design The mice were randomly assigned into 3 groups (n=10): Control, MS+ Cuprizone-fed group, and Treatment group. Before experiments, animals were kept in plastic cages under controlled environmental conditions, then MS groups were fed cuprizone  mixed into animals’ normal diet for six weeks to induce toxic-demyelination. In order to investigate LINGO-1 antibody effects on remyelination and neurobehavioral deficit, LINGO-1 antibody (10 mg/kg) was injected (IP) in the third week . The animals, in the positive control group (CPZ-fed without treatment), were administered 0.9% NaCl. The cuprizone diet, continued until the end of the experimental period .Cardiac perfusionFor transcardial perfusion, mice were anesthetized by ketamine (80 mg/kg) and xylazine (10 mg/kg), IP at the end of the experimental period, and perfusion of phosphate-buffered saline (5 ml) and 4% neutral formalin was used to fix brain tissues. Brains were removed and placed in formalin (10%). Then, kept in 10% formaldehyde solution at ambient temperature for 24 hr. Processing and sectioning of brain tissuesThe brain was separated from the skull, postfixed in 4% paraformaldehyde, and cryoprotected by overnight incubation in sucrose 30%, at 4 °C for 1 to 2 days. Brain tissues were microdissected, dehydrated, and embedded in paraffin. Five-micrometer tissue sections were made in the corpus callosum using a Cryostat . The sections were stored at -20 °C until final staining.Immunofluorescence for detection of MBP, NF200, and BDNF The brain sections were washed 3x with PBS. Sections were incubated in blocking solution (consisted of 0.1% Triton X-100 (Sigma-Aldrich), 1% bovine serum albumin, and 5% normal serum in PBS) for 1 hr, and then incubated overnight at 4 °C with primary antibody: anti-MBP , anti BDNF  and anti-NF200 . The day after, sections were rinsed with ice-cold PBS-tween and then incubated with the secondary fluorescence-labeled antibody for 1 hr at room temperature with goat Alexa Fluor 488 F (ab) anti-rabbit IgG as the secondary antibody at 1:1000 dilution. The sections were coverslipped and then images were captured from the stained frozen sections using a Labomed TCS 400 fluorescent microscope for consequent analysis. Brain images were quantified using the ImageJ software. Quantification was performed in three sections per mouse. The sections from all mice were immunostained at the same time. The semi-quantification of the intensities was evaluated using ImageJ software . The fluorescence signal profile was normalized by the background intensity. Areas related to the corpus callosum in one-half of the brain were analyzed for myelination intensity, BDNF, and NF200 intensity (%). Open field (OF) testThe open field test (OFT) is the most common method used to assess behavioral research in animal studies which evaluates the quantity and quality of exploratory behavior and activity of animals. For this purpose, the mice are placed into the center of an apparatus and multiple behavioral parameters such as distance moved, time spent moving and rearing are recorded over a set time (5 min). Some results  may indicate some aspects of emotional problems such as anxiety in mice . The opeBalance beam test (BBT)Measurement of motor coordination and balance can be useful to detect motor deficits caused by CNS lesions in rodents. This test can be more sensitive than Rotarod for some types of motor coordination deficits. At first, the animals are trained for 5 days before the initial assessment just to make sure that the mice’s behavior reflects motor coordination accurately during testing. Then, the balance state of the animals can be evaluated through the delay time to cross the beam. The purpose of this test was to assess the mice’s ability to maintain balance on a wooden beam (1 meter long) which was elevated 30 cm above the ground . A score of 2 was applied for navigating the beam with full weight support and keeping balance. If plantar placing of the paw was only partly possible, the animal scored 1.5. A score of 1 was given if the animal could cross the entire beam but without plantar placing of the hind paws. When only half of the beam could be crossed, a score of 0.5 was assigned, and for complete inability, a score of 0 was applied , 35. BeaStatistical analysisP<0.05). For all graphs, statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Statistical analysis was performed using GraphPad Prism 4 and SPSS 18 software packages. One-way analysis of variance ANOVA followed by Tukey’s post-test was used to compare the results of the study groups. Results were expressed as mean ± SEM. Differences were considered statistically significant when at least a 95% confidence level was achieved . Furthermore, the results showed that the level of MBP in the CPZ+ LINGO-1 antibody group was significantly higher than that in the CPZ group (P<0.001). MBP is a protein that is responsible for the multi-lamellar structure of myelin . MBP is PZ group , P<0.001LINGO-1 antibody promotes NF200 level in the corpus callosum of CPZ-fed miceP<0.001), and after treatment with LINGO- 1 antibody were increased significantly (P<0.001). These results showed that LINGO-1 administration might be effective in improvement of demyelination disease. To evaluate the number of axons in the corpus callosum, we used immunofluorescence staining for NF200. The neuro-filaments are some of the major components of the neuronal cytoskeleton involved in providing structural support for the axon and essential for axonal network formation . In corpficantly , P<0.001LINGO-1 administration significantly increased BDNF level in the corpus callosumP<0.001) compared with the control group. The results showed that the level of BDNF in the treatment group was significantly higher than in the CPZ group (P<0.001). BDNF concentrations were evaluated in the brain tissue of experimental groups to compare the LINGO-1 antibody effects on BDNF levels in the corpus callosum. The results showed a significant decrease in the brain BDNF levels of mice in the CPZ group . P<0.001PZ group . P<0.001LINGO-1 antibody administration improved behavioral testP<0.001) and the number of rears (P<0.01) in OFT. In the CPZ group, the number of squares crossed and the number of rears decreased compared with that in control mice fed normal chow . Furthermore, the mice in the cuprizone-fed group showed a lower score in the beam test at week 6, and the marks improvement was seen at the end of the study in the treatment group. With the balance beam test, we investigated the ability of mice to navigate across a 1-m-long beam. According to our data, there was a significant difference between experimental groups at the end of the experimental period (Therefore, it can be concluded that LINGO-1 injection can improve the beam test score in the CPZ model of demyelination. The beam’s ability to walk indicates the descending movement control of the vestibulospinal tract. The previous study has indicated persistent deficits in balance beam test at early stages of EAE  that proIn our study, we found that the use of the LINGO-1 antibody improved remyelination and behavioral changes in the CPZ model of demyelination in mice. The results showed that systemic administration of LINGO-1 antibody improved animal activities maybe through remyelination enhancement. In addition, the increased levels of MBP, NF200, and BDNF were seen in the corpus callosum after the three-week LINGO-1 antibody treatment. Taken together, our study has shown that LINGO-1 antibody may be effective in remyelination and neurobehavioral defects in MS.


Pseudomonas aeruginosa acyl-homoserine lactone quorum sensing (QS) can activate expression of dozens to hundreds of genes depending on the strain under investigation. Many QS-activated genes code for extracellular products. P. aeruginosa has become a model for studies of cell-cell communication and coordination of cooperative activities, which result from production of extracellular products. We hypothesized that strain variation in the size of the QS regulon might reflect the environmental history of an isolate. We tested the hypothesis by performing long-term growth experiments with the well-studied strain PAO1, which has a relatively large QS regulon, under conditions where only limited QS-controlled functions are required. We grew P. aeruginosa for about 1000 generations in a condition where expression of QS-activated genes was required, and emergence of QS mutants was constrained and compared the QS regulons of populations after 35 generations to those after about 1000 generations in two independent lineages by using quorum quenching and RNA-seq technology. In one lineage the number of QS-activated genes identified was reduced by over 60% and in the other by about 30% in 1000-generation populations compared to 35-generation populations. Our results provide insight about the variations in the number of QS-activated genes reported for different P. aeruginosa environmental and clinical isolates and, about how environmental conditions might influence social evolution.In the opportunistic pathogenic bacterium  Pseudomonas aeruginosa activates expression of a battery of genes by using acyl-homoserine lactone (AHL) quorum sensing (QS). There are two P. aeruginosa AHL QS circuits, the LasR-I and RhlR-I circuits. Genomic sequencing shows the genes for these circuits are highly conserved among hundreds of P. aeruginosa isolates from different environments (lasI gene codes for a N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) synthase, and lasR codes for a 3OC12-HSL-dependent transcription factor, which activates dozens of genes. The rhlI gene codes for a N-butanoyl-homoserine lactone (C4-HSL) synthase, and the product of rhlR is a C4-HSL-responsive transcription factor, which activates a set of genes overlapping those activated by LasR  were used to inoculate five tubes of a minimal broth containing only casein and adenosine (CAB) as carbon and energy sources. After inoculation, cells were transferred to fresh CAB daily for the next 160 days  results in emergence of cells, which show improved growth on adenosine resulting from a genome amplification of a region containing the LasR-dependent nuh gene , and a T515C single-nucleotide polymorphism (SNP) in lineage E. This finding was consistent with the idea that the populations were adapting to growth on casein. We asked if there was also adaptation to growth on adenosine. When transferred to a minimal medium containing only adenosine as a carbon and energy source, day-5 populations of both lineages grew slowly, but day-50 populations grew more rapidly . Therefore, we assessed growth yields by using plate counting to determine cell yields after 18 h in CAB for populations isolated after 5, 20, 50, 80 and 160 days, and found that there was a large increase in yield at day 50 , in which LasR mutants were not detected because examination of these lineages allowed us to address the question of how QS regulons might differ in size and composition in isolation from the complication of a QS mutant cheater subpopulation. Early in the experiment at day 5,  rapidly , as woult day 50 . This codenosine . There wdenosine . BecausepvdD and pvdJ, genes required for synthesis of the iron siderophore pyoverdine (nuh (two copies in one isolate and three in the other). One of the two lineage D isolates had two copies of a 90-kb nuh-containing region and the other isolate did not have amplification of a nuh-containing region .–24.pvdD 10.1128/mbio.00161-22.1TABLE S1Table S1, PDF file, 0.2 MB.Mutations in day-160 isolates from lineages D and E. Download Copyright © 2022 Smalley et al.2022Smalley et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa QS regulon has been gained by transcriptome analysis using QS gene deletion mutants . We determined levels of 3OC12-HSL and C4-HSL in LB culture fluids of lineages D and E of 5-day and 160-day populations at an OD of 2.0. In all cultures without AiiA, regardless of lineage or days of CAB passage, we found similarly high levels of 3OC12-HSL . C4-HSL levels were similar in both day-5 lineages ; however, day-160 populations produced less C4-HSL  than did Day-5 populations. To eliminate the possibility that variability in signal production might affect results of the transcriptomics experiments we added AHLs (2 μM 3OC12-HSL and 10 μM C4-HSL) to cultures of populations grown without AiiA lactonase. We note that these additions are similar to those used by others  higher levels in populations grown with added AHLs compared to populations grown with added AiiA lactonase were considered QS activated. This fold change cutoff was selected as it captured a previously determined core QS regulon (hcp secretion island III (HSI-3) cluster of type VI secretion system genes (PA2361-PA2371) in lineage D were classified as QS-activated, whereas only four of these genes in lineage E were classified as activated. Of the 137 QS-activated genes common to both lineages  or of the constituents of casein .It is possible that the observed loss of genes from the QS regulon could reflect a more global pattern of gene expression, not specific to the QS regulon. That perhaps there was a general lowering of the expression of genes not relevant to growth in CAB. To address this possibility, we compared gene expression in populations grown with AHLs from day 160 versus day 5. Of the 5570 PAO1 genes , we foun10.1128/mbio.00161-22.2TABLE S2Table S2, PDF file, 0.1 MB.Genes with decreased expression at day 160 in CAB-evolved Populations D and E, compared to day 5. Download Copyright © 2022 Smalley et al.2022Smalley et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00161-22.3TABLE S3Table S3, PDF file, 0.1 MB.Genes with greater expression at day 160 in CAB-evolved Populations D and E, compared to day 5. Download Copyright © 2022 Smalley et al.2022Smalley et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the lasI and rhlI, are retained in the day-160 QS regulons of both lineages  and two lectin genes (lecA and lecB), that may provide benefits in myriad environments, but not in CAB.There were 51 genes lost from the QS regulons of both populations D and E . There wThe elimination of a given gene from the QS regulon by day 160 in CAB could have many possible explanations. For example, there can be a mutation in the promoter for that gene, there can be a delay in the QS activation of that gene such that it falls below the cutoff for activation at an OD of 2, there could be a mutation in a co-activator of that gene, or there could even be a mutation in that gene. Some of these possibilities have long-term consequences, and others can perhaps be more readily reversed if conditions change.pqsA in our four day-160 clones used for genome sequencing . We selected pqsA because there was no mutation in its promoter region in either lineage, but there were variants in pqsR , the LasR-regulated gene encoding the transcriptional activator of pqsA  would affect the constitution of the QS regulon, and our results show that the regulon was substantially reduced. Our studies on P. aeruginosa can be put in the context of the rich literature on laboratory evolution of Escherichia coli, which has been used as a model to investigate long-term evolution over a span of tens of thousands of generations in a specific growth environment. For E. coli subjected to daily transfers to a fresh medium containing a single carbon and energy source, population fitness gains occur over time and certain mutations can sweep through a population  on casein and adenosine together, QS mutants are constrained. Our interpretation of this constraint is that LasR mutants can benefit from proteases produced by their parents, but they have lost access to adenosine  as the sources of carbon and energy. Protease-negative mutants were constrained in all five of the lineages we maintained for at least 50 days. By day 80, protease-negative mutants had broken through in two of the five lineages . We specividuals . These rividuals  and breaE. coli, fitness improvements were most dramatic early  and became progressively smaller over time  the number of QS activated genes at day 160 was only 26% of the number at day 5, and in the other population (E) it was 53%. One way to eliminate a gene from the QS regulon is by deletion, but our RNA-seq results and our genome sequencing of two isolates from each population indicated this was not a common event. There were only three deletions identified in the analysis, compared to hundreds of SNPs. It appeared that almost all QS regulon genes were intact. Their loss from the QS regulon could result from any of a several mechanisms, including delay in QS gene activation, modification of a coregulatory pathway, and subtle changes in levels of other transcriptional regulators in cells. Regardless of the mechanism, this seems to represent a flexible solution to reducing the cost of QS when P. aeruginosa reduced its QS regulon during long-term growth in CAB we focused on pqsA, a gene that showed greater than 10-fold QS activation in either day-5 population but not in either day-160 population . The pqsA promoter is activated by the PqsR transcription factor together with PQS, and pqsR transcription is activated by LasR. We found a point mutation in the two day-160 lineage E isolates, and this mutation was in fact fixed in the day-160 population. This presents a solution to the question of how the pqs operon is removed from QS activation and silent. In this solution, the pseudogene leaves an inactive copy of pqsR. It is conceivable that Pqs production can be recovered by repair of the pseudogene or expression of the pqs operon can become independent of PqsR. The two population D isolates showed different pqsA expression patterns in pure culture. One exhibited a low level of pqsA expression and the other showed delayed expression. Neither had a pqsR mutation. This population developed a different strategy, yet unknown, to release pqsA expression from QS activation. This finding is consistent with our other results indicating the populations are heterogeneous.To begin to understand ways in which pulation , Fig. 3BP. aeruginosa isolate might provide information about the ecological history of that isolate. We note that some of the differences between day-160 lineages and between day 5 and day 160 in a given lineage might be due to the noise inherent in RNA-seq analyses. Nevertheless, during our experiment, there was a reduction in the number of genes activated by QS. The increased population fitness observed between day 50 and day 160 in our metapopulations . Screening for protease-negative colonies was on skim milk agar plates as described elsewhere . The purified DNA was used as the template for PCR . Reads were aligned to the PAO1 reference genome (accession NC_002516) using StrandNGS version 3.3.1 . Variant analysis was performed with StrandNGS SNP, CNV and SV pipelines. We defined a SNP as having a variant read frequency of >90% and an indel as being <100 bp. Previously, we sequenced the genomes of two individual isolates of our laboratory PAO1 strain, MPAO1 , and we used this sequence for comparisons for SNPs calls and copy number/indel analyses.E. coli as described elsewhere  and stored at −80°C until use. AiiA was added to cultures at a final concentration of 100 μg/mL, which was sufficient to reduce 3OC12- and C4-HSL levels to below detection as measured by bioassay  fusion from recombinant lsewhere  except tbioassay .Scrapings from glycerol stocks of populations D and E (from day-5 and day-160 passages) were used to inoculate 3 mL of LB. After an overnight incubation, cells from these cultures were used to inoculate 3 mL of LB with a starting OD of 0.01. These cells were grown to logarithmic phase (OD 0.1–0.4) and then used to inoculate 25 mL of LB in a 250-mL baffled flask (starting OD 0.005) and one of the following was added: AiiA lactonase (100 μg/mL), AHLs , or no additions. Two biological replicates were prepared for each condition. At an OD of 2.0, AHL signals were solvent extracted from 5 mL of culture and measured by bioassay as described previously . At the 2 scale). Normalized transcript counts  to the earliest match location in the genome. Thus, although phenazine synthesis is QS-regulated, we excluded phzA1-G1 and phzA2-G2 from subsequent analyses of our differential expression data.RNA-Seq library preparation, bacterial Ribo-Zero rRNA depletion , and sequencing was performed by Genewiz . Library samples were divided into two separate HiSeq3000 runs, each with paired-end 2 × 150 bp read lengths. Trim Galore!  was used to trim adapters prior to alignment against the PAO1 reference genome (accession NC_002516) using StrandNGS version 3.3.1 . DESeq2  was usedt counts  were obtt counts . Note thpqsA and a promoterless gfp control , diluted to an initial OD of 0.01 in CAB or LB as indicated, and dispensed in 200 μl volumes microtiter dish wells. Fluorescence  and absorbance (OD600) was measured every 15 min for 14 h using a Biotek Synergy H1 microplate reader. Background fluorescence was determined using the promoterless pBBR-gfp control.We created a promoter probe fusion for  control  by usingassembly . PlasmidRaw sequencing reads for variant analyses of the two individual isolates from each of the day-160 lineages (BioSamples SAMN16400702-SAMN16400711) were deposited in the NCBI Sequence Read Archive under BioProject PRJNA667949. Raw sequencing reads and count matrices for transcriptome analyses of CAB-evolved populations D  and E  were also deposited under GEO accession GSE176411.


The fabricated device exhibited a threshold voltage (Vth) of 0.13 V and a maximum transconductance (gm) of 484 (mS/mm). At 38 GHz, an output power density of 3.22 W/mm with a power-added efficiency (PAE) of 34.83% were achieved. Such superior performance was mainly attributed to the high-quality Al2O3 layer with a smooth surface which also suppressed the current collapse phenomenon.In this work, a low-power plasma oxidation surface treatment followed by Al The quest for a high-capacity 5G network for increased data transmission requires wide bandwidth and high signal-to-noise ratio (SNR) to maintain the quality of service (QoS) of the system. Since the spectrum below 20 GHz has been crowded with commercial applications, allocation of bandwidth wide enough to accommodate the required channel capacity is almost impossible. As a result, operating at millimeter-wave frequencies has been the only solution for next-generation communication systems targeting at multi-giga-bit data transmission with minimum latency. In comparison with Si- and GaAs-based transistors, GaN-based HEMTs exhibit superior performance at high frequencies in terms of power density and efficiency due to the characteristics of the wide bandgap, high electron mobility and large breakdown field of GaN material. Successful demonstrations of the depletion-mode (D-mode) devices have been reported in ,2,3.T) and maximum oscillation frequency (fmax) of 6.0 and 9.8 GHz by adopting a gate-all-around structure in [Other than the D-mode devices, the E-mode devices exhibit certain advantages in terms of practical operations at system level. Since the devices require only positive supply, the complexity of the biasing network can greatly be reduced. Moreover, elimination of the negative supply also helps the suppression of the system noise. In the past, several approaches were reported for the development of E-mode devices. Fluorine plasma treatment was applied to insert negative charges into the barrier layer, resulting in a positive shift with the threshold voltage as a depletion region was formed in the 2DEG channel. A maximum output power density of 3.6 W/mm and peak PAE of 42% were measured at 18 GHz from the E-mode AlGaN/GaN heterojunction FET (HFET) [cture in .T and fmax in [2 treatment [6 plasma treatment, the gate leakage and pulse I–V characteristics were improved effectively due to the reduction in the amount of carbon on the semiconductor surface [2O3 dielectric layer under the gate region was adopted to suppress the hydrogen-induced weak bonds for leakage current reduction in [Gate recess is the major technology for the realization of the E-mode devices ,7,8. The fmax in ,13,14,15 fmax in , wet che fmax in , plasma  fmax in , descum  fmax in  and O2 treatment  which ha surface . Besidesction in . The fab2O3 layer. Compared with hydrochloric acid cleaning, a better surface morphology was achieved after the adoption of the wet surface treatment. Moreover, the imperfect layer of the gate region surface which included the native oxide and the bombardment damage caused by the plasma etching process could be effectively removed. Improvement in the device performance was observed with such surface treatment due to the suppression of the current collapse phenomenon. The maximum output power density and peak PAE were measured to be 3.22 W/mm and 34.83%, respectively, at 38 GHz.In this work, we fabricated an enhancement-mode AlGaN/GaN metal-oxide-semiconductor high-electron-mobility transistor (MOSHEMT) using the wet surface treatment technology followed by the deposition of a high-quality Al0.26Ga0.74N barrier layer. Room temperature Hall effect measurement exhibits a sheet carrier density of 9.7 × 1012 cm−2, a sheet resistance of 324 Ω/□, and an electron mobility of 1980 cm2/V·s, respectively. 2 ambient using a Premtek RTP-T41 rapid thermal annealing (RTA) system. A 25 nm-SiNx was deposited by STS310PC plasma-enhanced chemical vapor deposition (PECVD) with in-situ nitrogen plasma treatment prior to the film deposition [x film could reduce the dangling bond on the surface and act as hard mask at the recess region. The 150 nm gate foot was defined by using a 50 kV JEOL e-beam lithography (EBL) system (JBX 6000 FS) and the gate region was opened by fluorine-based ICP dry etching to remove SiNx film. Next, the gate recess process was performed using Cl2-based plasma with the following parameters: 40 sccm flow rate, 0.1 Pa pressure, and 200 W of RF power with 5 W of bottom bias. O2 plasma was performed by the ICP system  with low RF power of 50 W and the 5 W for the bottom bias. Afterwards, a thin oxide layer was formed on the surface. Then, the oxide layer was removed by wet surface treatment using NH4OH solution for 5 min. A 7 nm Al2O3 insulator was deposited by a Fujitec G2 atomic layer deposition (ALD) system  immediately after the surface treatment. The growth rate was 1 Å/cycle for 70 cycles and the temperature was kept at 250 °C. Afterwards, the active region was defined by nitrogen ion implantation. With the use of the ZEP/GL-2000 photoresists, the T-shape gate was formed as shown in x layer was deposited by PECVD after Ni/Au gate metal deposition and the lift-off process. The recessed depth of the gate region was approximately 4 nm. Finally, Ti/Au was deposited as an interconnect metal. The process flow is shown in Device fabrication started with the wafer cleaning by dipping in the acetone and isopropanol solution to remove the organic particles or contaminations. The ohmic contact consisted of a Ti/Al/Ni/Au metal stack deposited by ULVAC EVA650 E-gun evaporation system . Ohmic metal alloying was then formed at 820 °C for 30 s in Nposition . The SiNd and Al 2p core-level spectra of the control sample with HCl surface treatment and the sample with wet surface treatment, respectively. The Ga 3d and Al 2p peaks were separated into four main peaks Ga–O, Ga–N, Al–O and Al–N bonds. It is obvious that the intensity of the Ga–O and Al–O with a larger binding energy are much lower in the experimental sample. In other words, the Ga–O and Al–O bonding proportion decreased at the surface region. Such an effect indicates that with the application of wet surface treatment, the imperfect layer can be effectively removed. In [X-ray photoelectron spectroscopy (XPS) was adopted to investigate the surface chemical composition after applying the wet surface treatment. Generally, an imperfect layer caused by the environment pollutants, the native oxide, and the bombardment damage existed after the gate recess process. The existence of such a layer would degrade the device characteristics. It is thus of crucial importance to address this issue especially for devices at millimeter-wave frequencies. oved. In , the XPS2 devices with surface treatment exhibits a lower maximum drain current of 859 mA/mm compared to the devices without surface treatment, which has a maximum current of 931 mA/mm at VG = 2 V. After the wet surface treatment, a peak extrinsic transconductance (gm) 484 mS/mm and a threshold voltage (Vth) of +0.13 V were achieved at VDS = 5 V due to the removal of the surface oxidation layer by wet recess. While scaling down the distance between gate and channel, higher gm and lower current density were obtained for E-mode device. As for the gate leakage current for both cases shown in GS0, VDS0) = , the device with wet surface treatment shows minor collapse ratio of 3.7% which is much smaller than that of 17.78% for the one without treatment. It is obvious that the plasma damage caused by the dry etching process can be suppressed after the combination of the oxidation and wet etching treatment. The drain lag ratio has also decreased from 17% to 12.58% at VGS = 1 V and VDS = 5 V under the condition  = . Improvement in the current collapse phenomenon has been identified from both drain and gate lad measurements. It is worth mentioning that the drain lag results are owing to the deep traps in epitaxial layers, such as GaN iron-doped buffer [The measured DC characteristics of the thin barrier E-mode AlGaN/GaN HEMTs is shown in d buffer ,23. ThesT) and the maximum oscillation frequency (fmax) were 46/60 GHz and 109/100 GHz for the device with/without surface treatment. We have used the small-signal equivalent circuit using the procedure in [gs + Cgd) for devices without and with surface treatment are 98.95 fF and 86.7 fF, respectively. Thus, the higher cut-off frequency (fT) of the device with wet surface treatment can be attributed to the lower gate capacitance. However, slight degradation in fmax for the device with surface treatment is possibly due to the decrease of the output current and the lower 2DEG density.The small-signal RF S-parameters were measured from 100 MHz to 40 GHz using a Keysight N5227B network analyzer. A standard short-open-load-through (SOLT) calibration method was adopted to calibrate the system with the reference planes set at the tips of the corresponding probes. edure in  to extraj24.12) Ω and (92.15 + j126.12) Ω and those for the device with treatment are (35.85 + j28.31) Ω and (226.07 + j216.15) Ω, respectively. Under these conditions, a maximum output power density of 2.1 W/mm with a linear power gain (Gp) of 8.2 dB and a PAE of 22.84% were obtained at VDS = 10 V for the device without wet surface treatment. On the other hand, with the suppression of current collapse, a higher maximum output power density of 3.22 W/mm and a PAE of 34.83% were achieved for the device with surface treatment. Such a substantial improvement in the performance at Ka-band is mainly attributed to the suppression of the current collapse issue [DS. In all, the experimental results of the thin barrier E-mode AlGaN/GaN HEMT on Sapphire shows a great potential for low power consumption and low-cost power amplifier application.The large-signal performance was characterized using an on-wafer load-pull system at 38 GHz under continuous mode excitation. se issue . The mea2O3 as the gate dielectric layer. After the dry etching process and wet etching treatment, the damaged surface layer was removed. Moreover, the current collapse was eliminated due to the reduced trapping effect. Compared to a device without surface treatment, substantial improvement in the radiofrequency (RF) performance was achieved. The device exhibited a superior power performance, including a maximum output power density of 3.22 W/mm and a peak PAE of 34.83% at 38 GHz. These results demonstrate an attractive technique for fabricating high-performance E-mode HEMTs for power amplifier applications at millimeter-wave frequencies.A high-performance thin barrier enhancement-mode AlGaN/GaN HEMT was fabricated with wet surface treatment and high-quality Al


Among different approaches to the search for novel—safer and less addictive—opioid analgesics, biased agonism has received the most attention in recent years. Some μ-opioid receptor agonists with G protein bias, including SR compounds, were proposed to induce diminished side effects. However, in many aspects, behavioral effects of those compounds, as well as the mechanisms underlying differences in their action, remain unexplored. Here, we aimed to evaluate the effects of SR-14968 and SR-17018, highly G protein-biased opioid agonists, on antinociception, motor activity and addiction-like behaviors in C57BL/6J mice. The obtained results showed that the compounds induce strong and dose-dependent antinociception. SR-14968 causes high, and SR-17018 much lower, locomotor activity. Both agonists develop reward-associated behavior and physical dependence. The compounds also cause antinociceptive tolerance, however, developing more slowly when compared to morphine. Interestingly, SR compounds, in particular SR-17018, slow down the development of antinociceptive tolerance to morphine and inhibit some symptoms of morphine withdrawal. Therefore, our results indicate that SR agonists possess rewarding and addictive properties, but can positively modulate some symptoms of morphine dependence. Next, we have compared behavioral effects of SR-compounds and PZM21 and searched for a relationship to the substantial differences in molecular interactions that these compounds form with the µ-opioid receptor. The search for and development of novel opioid agonists have been recently intensified by the current opioid epidemic, which represents a major worldwide public health crisis. It has been shown that certain opioid agonists may preferentially activate one intracellular signaling pathway over another . This coAlthough the above-mentioned studies shed light on the general characteristics of SR agonists, the addictive potential of those compounds is still not fully explored. SR-14968 and SR-17018 were not tested in the same experimental models; therefore, the comparison of their effects is difficult.Here, we performed experiments allowing evaluation of the effects of SR-14968 and SR-17018  on physiAll experiments were performed according to the European Union regulations and the Directive 2010/63/EU and were approved by the II Local Bioethics Committee in Krakow . For all behavioral tests, C57BL/6J male mice were used. They were aged 8 weeks at the beginning of the experiments and weighed 25–30 g. Mice were group-housed, 10 per cage , under standard room temperature 22 ± 2 °C, humidity 50 ± 5%, and 12/12 h light-dark cycle, with free access to food and water . The starting size of each group was 10; however, some animals were excluded from the analysis, i.e., due to death/premature exclusion from the experiment because of poor health or biased preference during the preconditioning session of the conditioned place preference (CPP) test. The total number of animals as well as their suffering was minimized, according to the 3R principle.SR-14968 and SR-17018 were synthesized according to the previously published procedure  11]. Mo. Mo11]. Antinociception to thermal nociceptive stimuli was measured using tail flick and hot plate tests as described in more detail in our previous work . Basal rIn the tail flick test , the light beam was applied to the dorsal side of a mouse’s tail, and the time latency to tail withdrawal or shaking was recorded. To avoid tissue damage, a cut-off latency was set at 9 s.The hot plate test was carried out using a hot/cold plate apparatus . Mice were placed on a plate heated to 52.5 °C, and the time latency to the first sign of spinally mediated withdrawal reflexes (a paw flinching) as well the first sign of complex behavior (licking/biting of the paw and/or jumping) were measured. A maximum exposure time was set at 30 s (cut-off) to avoid tissue damage. Both responses were expressed as % MPE calculated according to the formula: [(T1 − T0)/(T2 − T0)] × 100, where T0 and T1 are the response latencies before and after drug injection, respectively, and T2 is the cut-off time.To study the effects of SR agonists on morphine-induced antinociception, mice were pretreated with SR compounds 30 min prior to morphine, and then antinociception was measured using the tail flick test.To assess the development of tolerance to antinociception induced by SR agonists, mice received injections with the drugs for 7 consecutive days and were tested in the tail flick assay 1 h after drug administration.In another experiment, aimed to test the influence of SR compounds on the development of tolerance to morphine antinociception, mice were pretreated with SR compounds 30 min prior to morphine, and then the tail flick test was performed.Physical dependence on the SR compounds was measured using a paradigm described previously . Mice weIn the second experiment, the influence of SR compounds on physical dependence to morphine was tested. To induce dependence, mice were chronically treated with increasing doses of morphine for 4 days as previously reported . BrieflyThe severity of withdrawal was presented as a number of jumps considered as the most prominent symptoms as well as the overall withdrawal score calculated according to the formula described in detail previously .The CPP test was performed as described in our earlier work . BrieflyThe measurement of locomotor activity lasted for 6 days and was performed using activity chambers (custom-made). Each day, all animals were placed in a chamber for 30 min, and after this time, they received the appropriate drug injection and were placed back in the boxes for an additional 2 h. The expression of locomotor activity was tested after 8 days—the incubation period on day 14. All animals were habituated to the chambers for 2 h, 1 day before the onset of the experiments.All examined compounds, PZM21, SR-17018, and SR-14968 (both isomers), were docked to the crystal structures of the µ-opioid receptor of the following PDB codes: 4DKL, 5C1M, 6DDF. The compounds were prepared for docking with the use of LigPrep  from thet-test .Statistical analyses were performed using GraphPad Prism . For the data analysis, we performed an unpaired two-tailed t-test f, a one-t-test b,c, Krust-test c or two-t-test a. Post-h = 3.169, p = 0.0005,  = 12.36, p < 0.0001,  = 18.68, p < 0.0001, At the beginning of the study, we performed tail flick and hot plate tests to assess the antinociceptive effects of SR-14968 and SR-17018. Treatment with both compounds resulted in long-lasting and dose-dependent antinociception in the tail flick test (SR-14968: interaction effect (treatment × time) F = 14.51, p < 0.0001, paw licking/jumping F = 13.86, p < 0.0001,  = 6.385, p = 0.0014, paw licking/jumping F = 10.77, p < 0.0001, (18) = 2.899, p = 0.0096, paw licking/jumping t(18) = 0.9240, p = 0.3677, In the hot plate test, we distinguished two types of reactions to nociceptive stimuli: paw flinching and licking of the paw or jumping. Administration of SR compounds increased the time latency to both types of reaction in a dose-dependent manner  of this compound administered twice daily would cause a significant deterioration of animal well-being. The single injection of 0.3 or 1 mg/kg of the SR agonist was used for the assessment of the influence on morphine antinociception and tolerance, respectively, and the effect of 1 mg/kg on the naloxone-precipitated morphine withdrawal . = 5.463, p < 0.0001, In the next part of the study, we assessed the physiological symptoms of addiction—tolerance to antinociception and precipitated withdrawal syndrome in mice dependent on SR compounds. The results showed that repeated administration of SR agonists led to tolerance. However, when compared to morphine, it developed more slowly, as morphine-induced tolerance was observed on day 3, while such an effect in SR compound-treated groups was noticed on day 5 or 7 (interaction effect (treatment × time) F = 65.99, p < 0.0001, (3) = 31.33, p < 0.0001,  = 38.18, p < 0.0001). Therefore, our results indicate that regardless of their G protein bias, SR compounds, especially SR-14968, induce profound physical dependence in mice.Chronic treatment with SR compounds resulted also in physical dependence, as measured by naloxone-precipitated withdrawal signs  = 4.266, p = 0.0119, Next, we determined the influence of SR compounds on subjective aspects of addiction-like behavior. We performed a CPP procedure to assess the impact of SR-14968 and SR-17018 on reward-associated memory. The data obtained indicate that treatment with both SR compounds and morphine had rewarding effects in this test  = 11.29, p < 0.0001,  = 2.583, p = 0.0006, We then measured animals’ locomotor activity over 6 consecutive days to test whether SR compounds cause sensitization/tolerance of locomotor effects. On the first day of the experiment, the acute effects of the tested compounds on locomotion were assessed. The results showed that morphine- and SR-14968-treated mice presented enhanced locomotor activity when compared to control animals  = 6.093, p = 0.001,  = 1.514, p = 0.0853, The expression of locomotor activity was measured after an 8-day incubation period. The data show that only the treatment with SR-14968 resulted in a significantly increased expression of locomotor effects (interaction effect (treatment × time) F = 5.17, p = 0.0136); however, the analysis showed no statistically significant interaction effect ((treatment × time) F = 0.7646, p = 0.6347, Finally, we aimed to assess the influence of SR agonists on certain behavioral effects of morphine. We tested the impact of SR compounds on morphine-induced antinociception. Moreover, taking into account that SR compounds present prolonged action and, when compared to morphine, tolerance to their antinociceptive effects develops more slowly, we aimed to test their ability to modulate morphine-induced tolerance and physical dependence. SR agonists or vehicle were administered 30 min prior to morphine injection, and antinociceptive responses were measured using the tail flick test. We used the dose of 5 mg/kg of morphine to evoke a moderate antinociceptive effect and the lowest doses of SR compounds, as those agonists had strong antinociceptive action. There is a tendency toward enhanced antinociception in mice cotreated with SR agonists and morphine  = 1.459, p = 0.2026,  = 12.82, p = 0.0001).We then tested the effects of SR agonists on the development of morphine-induced antinociceptive tolerance. The drugs were administered 30 min prior to each morphine injection (days 1–7). The data analysis showed no interaction effect ((treatment × time) F = 10.69, p = 0.0005,  = 2.434, p = 0.1091). SR-14968 slightly attenuated withdrawal; however, no statistically significant changes were observed.In the next experiment, we tested the influence of SR agonists on the severity of withdrawal symptoms in mice chronically treated with escalating doses of morphine by administration of SR compounds 30 min prior to naloxone. Pretreatment with SR-17018 resulted in a decrease in the number of jumps . As SR-14968 was experimentally tested as racemate, both its isomers were modeled independently.The obtained docking poses are presented in 2×63 and W135, as well as two residues from the 6th transmembrane helix (TM6) and lack of contact by PZM-21 with V1453×28 and I1463×29.The docking results indicate that although (R)-SR-14968 adopts a slightly different orientation of the piperazine moiety in 4DKL crystal structure, the set of amino acids interacting with this ligand in the receptor binding site is similar to the set of residues, which form contact with SR-17018 and PZM-21. The most visible differences in the ligand-protein interaction matrices referring to the 4DKL-based dockings involve lack of interaction by (S)-SR-14968 with N129For 5C1M, which represents an activated form of the µ-opioid receptor, it was SR-17018 that slightly shifted the orientation of the piperidine and chlorophenyl moieties in comparison to PZM-21 and both isomers of SR-14968. It is reflected in the ligand-interaction matrix as a lack of interaction by SR-17018 with TM7. The most consistent poses of all SR agonists examined were obtained in the 6DDF-based dockings, with only slight differences in contact patterns with the second extracellular loop (ELC2). In the case of experiments with 6DDF, it was PZM-21 that adopted a slightly different orientation in comparison to the rest of the compounds, and it was manifested mainly by more frequent interaction by PZM-21 with ELC2.In addition, for each ligand-receptor complex, the binding free energies were determined .The calculated binding free energies vary depending on the crystal structure used. For the inactive receptor form (4DKL), the highest binding energy was calculated for PZM-21 , and the lowest  for (S)-SR-14968. Interestingly, for this crystal structure, the highest differences between SR-14968 isomers were obtained . SR compounds were characterized by the highest binding energies for 5C1M crystal structure, whereas for 6DDF, it was again PZM21, for which the highest energy was supposed to be released upon binding. It should be remembered that such differences might be a result of slight variations in the conformation of the receptor binding sites for 5C1M and 6DDF, as 5C1M conformation is stabilized by the small molecule agonist BU72, whereas in the 6DDF, peptide agonist DAMGO was co-crystallized.As the set of amino acids differentiating examined compounds varied for different crystal structures used in the study, MD simulations for all of the obtained ligand-receptor complexes were carried out. Ligand-protein interaction diagrams, which enable detailed analysis of changes in the ligand-protein contact patterns, are included in the The ligand RMSD values clearly indicate that SR compounds (depicted in blue) are more stably fitted in the activated µ-opioid receptor  in comparison to PZM21 (red). On the other hand, the situation is reversed for the 4DKL crystal structure (which refers to the inactive receptor state) when atoms of PZM21 change their position to a lower extent than observed for SR compounds.In order to indicate amino acids that might be crucial for specific compound effects observed in behavioral studies, a detailed analysis of the frequency of ligand-receptor interactions during MD simulations was carried out. As we were mainly interested in finding discriminants between the SR compounds and PZM21, all data obtained for SR compounds were averaged and compared with the PZM21 interaction patterns. Differences in the frequencies of interactions during MD between SR compounds and PZM21 were determined for each amino acid of the target proteins, and positions with the highest discriminative power are gathered in 3×32 for 4DKL and Q1262×60 for 5C1M. Interestingly, Q1262×60 is a residue for which such high differences occur consistently for all crystal structures examined. In all cases, it was PZM21 that made contact with this amino acid more frequently in comparison to SR compounds. When only activated receptor forms are considered , there are 4 positions with high discriminative SR compound/PZM21 power: V2385×43, V3026×55, I1463×29, and Y3287×42; however, in this case, the tendencies in the contact preferences are not clear, as only contact with V3026×55 is preferred by PZM21 for both 5C1M and 6DDF. For other positions, the contact preferences varied depending on the crystal structure considered. There is one position (V1453×28) that is shared by 4DKL and 5C1M (with consistent contact preferences for PZM21) and 3 residues that are common for the 4DKL and 6DDF pair , with contact preference consistency occurring only for Y1503×33 (more frequent interaction with PZM21).Analysis of the interaction patterns obtained during MD simulations indicated that there is a quite extensive set of positions with significant differences in the contact frequencies between PZM21 and SR compounds. There are positions for which a difference of over 50% in the interaction frequency occurred during MD, such as D149In the present study, we showed that SR-17018 and SR-14968, highly G protein-biased μ-OR agonists with a wide therapeutic window, possess rewarding properties. Results obtained in the CPP test indicate that both agonists cause reward-associated behavior. It should be mentioned that a vehicle used for the preparation of solutions displayed some aversive properties in the CPP test. As both SR-agonists and morphine were dissolved in the same vehicle, we assume that SR-14968 and SR-17018 induced rewarding effects comparable to morphine. This suggests that G-protein biased μ-OR signaling mediates opioid reward and is in accordance with the results that morphine-induced place preference is enhanced or does not change in mice with disrupted β-arr2 functions ,26. On tWe also examined whether SR compounds affect the locomotor activity of mice upon both acute and repeated administration. We observed that SR-14968 induces a significant enhancement of acute locomotor activity of mice, but the continuation of treatment with this agonist results in toleration of the locomotor effects (between days 1 and 6). At the same time, no significant changes under both acute and chronic treatment conditions were found in mice from the SR-17018 and morphine groups. After the 8-day incubation period, a profound increase in locomotor activity (about 3-fold greater than in the previous measurement) was noted in animals treated with SR-14968. Again, no significant differences were found in the SR-17018 and morphine groups. Thus, this part of the study revealed a considerable distinction between the effects induced by SR-14968 and SR-17018. In our previous report, we showed that PZM21 did not change animals’ locomotor activity in the same experimental conditions as in the current study . Only inWe demonstrated that SR-14968 and SR-17018 exert a dose-dependent analgesia, which is in line with previous observations ,14. HoweIn the present study, we also showed that repeated treatment with both SR-14968 and SR-17018 results in antinociceptive tolerance. However, the development of tolerance was delayed, when compared to morphine. Previous research was aimed to determine the development of tolerance following chronic SR-17018 administration. Grim et al.  reportedThe results of the present study indicate that both SR compounds cause physical dependence, as we observed a naloxone-precipitated withdrawal syndrome in mice chronically treated with the compounds. SR-14968-treated mice presented a severe naloxone-precipitated withdrawal, even though this agonist was administered at a very low dose. It can be assumed, therefore, that it is a highly addictive compound. SR-17018 also caused signs of physical dependence, albeit less intense than SR-14968. Grim et al.  reportedFurther, our results indicate that SR compounds may influence some morphine-induced effects. SR agonists slightly potentiate morphine-induced antinociception. Co-treatment with morphine and SR agonists may slightly delay the development of tolerance, and pretreatment with SR-17018 can diminish the severity of naloxone-precipitated withdrawal. The results of our study are in line with the data presented by Bohn’s group . The autTo summarize, we demonstrated that SR-14968 and SR-17018 possess some addictive features. Both compounds induce comparable rewarding effects and cause lightly delayed development of antinociceptive tolerance. SR-14968 leads to more severe naloxone-precipitated withdrawal, indicating that it causes stronger physical dependence. Our observations bring a functional characterization of two agonists from the SR compound group and suggest that high G protein bias may not determine the nonaddictive properties of μ-OR agonists. In the context of the latest controversies regarding the G protein-biased opioids ,22,35,36Our data and several other sources of evidence  suggest 2×60, it is advisable to carefully examine contact with this amino acid during the design of new SR compounds/PZM21 derivatives. This position was also indicated in the recent work by Ricarte et al. [3×32 was indicated in our study for 4DKL-based experiments (PZM-21 preference) and in the work [3×33 ).The molecular modeling study did reveal in the μ-OR an important set of positions with significant differences in contact preferences and interactions with PZM21 and SR compounds. There was clear evidence that the stability of the compound orientation in the binding site of the activated form of μ-OR may play a role in the compounds’ intracellular signaling and functional activity. Increased stability of SR compounds in the activated receptor form is in line with the recent findings of Stahl et al. on SR-17018 and structurally related agonists  . Therefoe et al.  (where mthe work , it was Overall, the available data point out that G protein-biased μ-OR agonists may constitute a heterogeneous group with different opioid adverse effects and addictive potential. The complexity of μ-OR signaling requires an individual assessment of different aspects of behavior, taking into account other pharmacological attributes of the given agonist.To conclude, in the current work we showed that SR agonists, despite the G protein bias, still possess some addictive properties, as they induce tolerance and dependence and cause reward-associated behavior. At the same time, our results suggest that these compounds may be useful for the modulation of morphine dependence. The data obtained in studies on SR compounds suggest that G protein signaling appears to mediate reward. It is in contrast to the other G protein-biased opioid, PZM21. Molecular modeling did reveal that there is a set of amino acids of µ opioid receptor with significant differences in the contact preferences between PZM21 and SR compounds, which may suggest that other factors such as downstream effectors should be considered.


Functional lateralization of the prefrontal cortex has been implicated in stress and emotional disorders, yet underlying gene expression changes remains unknown. Here, we report molecular signatures lateralized by chronic social defeats between the two medial prefrontal cortices (mPFCs). Stressed mice show 526 asymmetrically expressed genes between the mPFCs. This cortical asymmetry selectively occurs in stressed mice with depressed social activity, but not in resilient mice with normal behavior. We have isolated highly asymmetric genes including connective tissue growth factor (CTGF), a molecule that modulates wound healing at the periphery. Knockdown of CTGF gene in the right mPFC by shRNA led to a stress-resistant behavioral phenotype. Overexpression of CTGF in the right mPFC using viral transduction induces social avoidance while the left mPFC thereof prevent stress-induced social avoidance. Our study provides a molecular window into the mechanism of stress-induced socioemotional disorders, which can pave the way for new interventions by targeting cortical asymmetry.The online version contains supplementary material available at 10.1186/s13041-021-00802-w. Stress induces numerous physiological changes in the brain, but only some individuals develop emotional disorders. Studies have sought to identify key mechanisms and molecules that explain the relationship between stress and emotional disorders. Brain imaging studies have revealed that patients with post-traumatic stress disorder (PTSD) , 2 or deAccording to the functional divergence hypothesis, the two mPFC hemispheres play different roles in stress perception and resilience. In mice  and ratsSeveral studies have sought to identify molecular effectors that mediate stress responses by exploring stress-induced changes of gene expression in the mPFC –13. CompC57BL/6 J male mice were used for all experiments. Before stress exposure, mice were housed in groups of 5 or 6 under a 12:12 h light:dark cycle  with food and water available ad libitum. All animal care and experimental procedures were performed in accordance with protocols approved by the Animal Care and Use Committee of Korea Advanced Institute of Science and Technology (KAIST).13 GC/ml titer) was delivered to the mPFC  using a Nanofil syringe with a 33-gague injection needle . As a control, AAV2/5-Scramble shRNA-CMV-mCherry-hGH  was injected into the same location. Overexpression of CTGF in the mPFC was achieved by delivering AAV2/9-CamKIIα-mCTGF-IRES-mCherry  to the mPFC; as a control, AAV2/5-CamKIIα-mCherry  was injected into the mPFC in the same manner. After surgery, mice were returned to their home cages and housed for 3 weeks to allow recovery and viral expression.Five-week-old mice were anesthetized with Avertin  and placed in a stereotaxic frame  for injection of viral vectors. For small hairpin RNA (shRNA)-mediated knockdown of CTGF, the viral vector AAV2/9-GFP-U6-mCTGF-shRNA  were screened for aggression as previously described . Each 8-The test for social avoidance was performed in a dark room in a white acrylic open-field box (42 × 42 cm) with a removable iron mesh cage (10 × 6.5 × 42 cm) that was used to secure the social target in the middle of one side of the box. Each experimental mouse was placed in the center of the open field and allowed to move freely for 150 s in the absence and presence of a social target . All mouse behavior was recorded under infrared lights using a digital video camera, and the time spent in the interaction zone was analyzed using EthoVision XT . The interaction zone was defined as previously described  . A sociaExperimental mice were subjected to a forced swim stress as previously reported . BrieflyThe sucrose preference test (SPT) was adapted from the previously reported 8-day sucrose preference protocol , which i2). Total RNA was prepared from each of the representative mice using the TRIzol reagent  and further purified using an RNeasy kit . RNAs were isolated from the left and right mPFC tissues of susceptible, resilient, and control mice and used for triplicate microarray analyses on a MouseRef-8_V2 Expression BeadChip .The raw microarray data have been deposited in the NCBI Gene Expression Omnibus at accession number GSE114224. Mice exposed to chronic social defeat stress were subdivided into two groups according to their sociability index (SI). The average of SI of mice for microarray analysis is 1.76 , 1.61 , and 0.5 . Non-defeated control mice were also selected from those who underwent a social interaction test . One day2 scale and normalized using the Bioconductor lumi package . The average SI values for non-defeat control, resilient, and susceptible mice were 1.5, 1.5, and 0.8, respectively. b. The SI values of mice that were selected for the microarray analysis. The average SI of the selected mice were 1.76 (n = 8), 1.61 (n = 8), and 0.5 (n = 7) for the control, resilient, and susceptible groups, respectively. c. Full list of the SI values of the mice used in the microarray analysis.Additional file 2: Figure S2. RT-qPCR confirmation of lateralized genes, categorized according to hemispheric dominance. Changes in the expression levels of left- and right-dominant genes were confirmed by RT-qPCR analysis. a. The Gapdh-normalized expression of the left-dominant genes, Cux2 and Wfs1, in the mPFC, presented as a bar graph. The expression levels of Cux2 and Wfs1 were higher in the left mPFC of susceptible mice, while the levels of these genes were similar in the left and right mPFC of non-stressed and resilient mice. b. The Gapdh-normalized expression of the right-dominant genes, Ctgf, Mbp, and Rprm, in the mPFC. The expression levels of Ctgf, Mbp, and Rprm were higher in the right mPFC of susceptible mice, while the levels of these genes were similar between the left and right mPFC of non-stressed and resilient mice. c. The left/right (L/R) expression ratios of the left-dominant genes, Cux2 and Wfs1, in the mPFC, presented as a bar graph. The ratios of Cux2 and Wfs1 were higher in susceptible mice than in non-stressed and resilient mice, whose ratios were similar. d. The L/R expression ratios of the right-dominant genes, Ctgf, Mbp, and Rprm, in the mPFC. The ratios of Ctgf, Mbp, and Rprm were lower in susceptible mice than non-stressed and resilient mice, whose ratios were similar.Additional file 3: Table S1. Analysis of DEGs in the two hemispheres of the mPFC in mice with social defeat stress versus non-stressed mice. Significant DEGs with a FDR adjusted p-value cutoff of 0.05 are shown. AveExpr, averaged expression of microarray genes; t, moderated t-statistic; B, B-statistic.Additional file 4: Table S2. List of 526 DEGs with FDR adjusted p-value cutoffs of 0.05 corresponding to genes presented in heatmap format in Fig. 1b according to their log2L/R values.Additional file 5: Table S3. Comparison of averaged expression values of DEGs between the two mPFC hemispheres, presented in dot plot format in Fig. 1c.Additional file 6: Table S4. The 526 genes exhibiting laterality listed, according to their log2L/R values. Laterality genes with positive values are expressed more highly in the left mPFC, and those with negative values are expressed more highly in the right mPFC. The genes used in rank order analysis in Fig. 1e.Additional file 7: Table S5. Differences in the expression of the housekeeping genes, Actb(b-actin), Gapdh, B2m .


SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load  which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups. Betacoronavirus [In late 2019, several cases of an atypical pneumonia were identified in China, which were linked to a novel coronavirus named as 2019 novel coronavirus, 2019-nCoV, and renamed as SARS-CoV-2 after identification of similarity to the severe acute respiratory syndrome (SARS) coronavirus. SARS-CoV-2 was then considered responsible for the coronavirus-associated disease (COVID-19). Its zoonotic origin was traced to bats due to its similarity with bat SARS-CoV-like coronaviruses, but it is speculated that an intermediate host also exists, at this moment pangolins being the most likely one [onavirus .https://www.worldometers.info/coronavirus/, accessed on 2 May 2021).The typical manifestations of COVID-19 are similar to those of SARS, which include fever, myalgia, dry cough, dyspnea, fatigue, and radiological evidence of ground-glass lung opacities compatible with atypical pneumoniae, but other symptoms differ, such as less frequent diarrhea compared to SARS . COVID-1https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas#individual-molecular). Even though all these methods are highly reliable and sensitive for the detection of SARS-CoV-2, as evidenced above, the high number of reported cases and the rapid spread have generated reagent shortages in certain locations, in particular, low-resource countries. This, in addition to the turnaround time needed to obtain an RT-qPCR result, has generated the need for the development of alternative methodologies suitable for population surveillance and fast clinical diagnosis. Among these, isothermal nucleic acid-based techniques have found a niche, and a plethora of methodologies have been reported recently [Due to this rapid dissemination of the virus, it is critical to accurately identify infected individuals in order to set up appropriate measures to limit further spread of the virus and so limit the disease and associated complications. The gold standard to perform the clinical diagnosis of the virus relies on its detection by reverse transcriptase real-time polymerase chain reaction (RT-qPCR) in nasopharyngeal swabs, for which several official protocols have been made publicly available, such as those from the CDC in the USA, the Institute Pasteur in France, or Charité in Germany ,6,7. Addrecently ,9,10,11.recently ,13, nakerecently ,15,16, orecently ,22.In the current study, the main goal was to develop a multi-target reverse transcriptase LAMP (RT-LAMP) assay for the detection of SARS-CoV-2. The performance of the methodology was evaluated against a panel of randomly collected clinical nasopharyngeal swabs, which were gathered by the Virology Service of the Portuguese Oncology Institute of Porto (IPO-Porto), and the results were compared against their gold standard RT-qPCR methodology.® VR-1986HK™, LGC Standards S.L.U., Barcelona, Spain), or with the Twist Synthetic SARS-CoV-2 RNA controls 1 to 6, as well as 14 and 15 . The batch of heat-inactivated virus acquired was provided at a final concentration of 3.75 × 105 genome copies/µL, while the synthetic controls were indicated to have 106 copies/µL. The synthetic controls 14 and 15 corresponded to the variant of concern (VOC) B.1.1.7. A rat coronavirus was used as negative control . RNA was extracted using the NucleoSpin Dx Virus kit  following the manufacturer’s instructions.For safety reasons, all the experiments performed in the evaluation of the methodology were performed either with the heat-inactivated SARS-CoV-2 virus (strain 2019-nCoV/USA-WA1/2020. ATCCN gene and the ORF3a using primerexplorer V5 (http://primerexplorer.jp/lampv5e/index.html). Primer design was based on the sequences of SARS-CoV-2 NC_045512 for each gene, and was obtained from NCBI. Details regarding the specific sequences are provided in Due to its novelty and promising results, the primers described by Mautner et al., targeting the ORF8 of SARS-CoV-2, were selected to be used in this study . In addiThe setup of the reactions for all three genes was performed by fluorescent RT-LAMP with the following components: FIP/BIP 800 nM, F3/B3 200 nM, and LF/LB 400 nM, 12 µL of Fast Fluorescent mastermix , 0.4 µL of OptiRT , 0.04 µL of ROX reference dye , and 3 µL of template . Ten-fold dilutions from the heat-inactivated virus were prepared in RNAse/DNAse-free water . The reactions were run at 65 °C for 30 min (the fluorescence data were acquired every 30 s over 60 cycles). The incubation was followed by a melt curve stage, which was performed by heating up the samples at 95 °C for 1 s, cooling down to 80 °C for 20 s, and heating again up to 95 °C, measuring the fluorescence over continuous increments of 0.015 °C/s. Having reached the final temperature, it was kept for 1 s. The reactions were performed in a QuantStudio 5 Real-Time PCR System with the QuantStudio™ Design and Analysis Software v1.4.3. .It is advised to avoid the opening of the reaction vessels, as LAMP is highly prone to false positive results due to amplification product cross-contamination. If this occurs, it will make it necessary to repeat the assays, thus delaying the results and increasing the cost of analysis ,26,27.6 down to 50 copies. Each dilution was analyzed in triplicate for each one of the genes, and the test was repeated three times, thus rendering a total of nine replicates per concentration. The results were modelled by probit analysis with MedCalc .The determination of the analytical sensitivity was performed using the synthetic control 1 as a reference. This control was ten-fold serially diluted from 10This study was approved by the Ethical Committee of IPO-Porto (232/020). Nasopharyngeal swab samples were collected from routine procedures for SARS-CoV-2 diagnosis at IPO Porto. After collection, samples were transported in sterile containers and delivered to the Virology Service in less than one hour, and stored at 4 °C until further processing. Samples were processed according to the safety instructions in a biosafety level 2 cabinet and inactivated with lysis buffer. The RNA extraction from clinical samples was performed with the reference protocol followed by the Virology Service of IPO-Porto, as detailed below.® StarMag Viral DNA/RNA kitSeegene  in a StarMAG System , followed by RT-qPCR amplification with the Allplex 2019-nCoV Assay  in a CFx-96 system from CFX96 Real-Time PCR Detection System . This assay targets four viral genes  and uses a total of 10 µL of template per sample. The results were analyzed using the Seegene Viewer software , considering positive samples if amplification was observed for the targets at cycle threshold (Cq) < 40 as per manufacturer’s instructions.The SARS-CoV-2 diagnostic at IPO-Porto was performed from 200 μL of sample with automated RNA extraction using the RNaseP by LAMP to rule out reaction inhibition [Nasopharyngeal swab samples collected at IPO-Porto were analyzed with the RT-LAMP assay and compared with the reference RT-qPCR protocol as previously described. With the RT-LAMP assay, a clinical sample was considered positive for SARS-CoV-2 if any of the selected genes were positive. The samples with a negative RT-LAMP, and with a delay in the internal control of the gold standard RT-qPCR, were tested for human hibition .In this sense, those samples providing positive results by both methods will be categorized as positive agreements (PA); likewise, those with negative results by both assays will be considered negative agreements (NA). If the RT-LAMP method reported a positive result while the RT-qPCR was negative, it would be classified as a positive deviation (PD), and if the RT-LAMP obtained a negative result with a positive RT-qPCR, it will be considered a negative deviation (ND).https://sample-size.net/.The categorized samples were used to calculate the diagnostic sensitivity, specificity, and accuracy  of the RT-LAMP method. In addition to these, the positive and negative predictive values (PPV and NPV) along with the Cohen’s kappa (k) were also calculated. These calculations were performed as previously published ,29, and https://blast.ncbi.nlm.nih.gov/Blast.cgi). Furthermore, the assay proved suitable for the detection of the VOC B.1.1.7. This variant is of great importance since it rapidly expanded in certain countries, and as of February 2021, accounted for 95% of the SARS-CoV-2 infections in England, and since then has been already identified in 82 countries [N [The three sets of primers correctly detected all the synthetic controls, along with the heat-inactivated human coronavirus, while no amplification was observed for the rat coronavirus. The specificity of the primers designed was further confirmed by BLAST analysis (ountries . All thetries [N .N with results ranging from 7 to 10 min (Tm 86.88 ± 0.20 °C) and the slowest was the ORF3a, reporting positive results for these controls in 10 to 14 min (Tm 86.39 ± 0.20 °C). The Tm values with their corresponding SD were experimentally determined based on the results obtained for all the positive samples analyzed. The gene reporting the fastest amplification was, on average, ORF8 as all the synthetic controls amplified in 5 to 8 min (average Tm 83.66 ± 0.17 °C), followed by 4 copies, but differences became apparent below this quantity. With ORF3a, the different replicates of all the dilutions were negative below 104 copies. Regarding ORF8, it was still possible to detect down to 100 copies but the detection probability dramatically decreased from 100% to 33% and 11% for the detection of 104, 103, and 100 copies, respectively. The most sensitive target was the N which was capable of detecting 103 copies with a detection probability of 66% and 100 copies with a probability of 22%. With the current conditions, none of the targets were capable of detecting 50 copies. The results obtained are in the same range of other previously published RT-LAMP studies, such as the one from Jang et al., which reached 101–102 copies/µL targeting RdRp, E, and N, or that of Mautner et al., who reported limited detection of 102 copies/µL [The analysis of the ten-fold dilutions of the synthetic control 1 indicated that all the targets were capable of reproducibly detecting the RNA down to 10opies/µL ,32. MinoA total of 152 clinical samples were analyzed in the current study. Attending to the reference RT-qPCR protocol, 49 were positive and 103 were negative. Regarding the multi-target RT-LAMP assay, 31 were positive and 102 were negative, 1 sample was classified as a PD and 18 as ND. These results are summarized in N, and 5 were only positive for N . Overall, the highest number of positive samples was obtained targeting the N gene, but solely targeting this gene could result in detection problems when thinking on novel variants, which are constantly being reported; thus, targeting more than one gene/fragment would be highly recommended to avoid this issue [N gene in their multiplex RT-LAMP assay implementing strand-displacement probes [Regarding the RT-LAMP, out of the 31 positive samples identified, 21 were positive for the three targets, 5 were positive for the combination ORF8-is issue . In thist probes . Some ofIn the evaluation of the RT-LAMP methodology, only 1 PD was observed out of the 152 samples analyzed, thus demonstrating its high specificity, which was calculated to be of 99.0%. Regarding the sensitivity, the results were not as good since 18 ND were obtained, rendering a sensitivity of 63.3%. This resulted in an overall efficiency of the method of 87.5%, a positive predictive value of 96.9%, a negative predictive value of 85.0%, and a Cohen’s k of 0.69. Nevertheless, this is interpreted as “good concordance” between the novel and the reference method. Similar results were reported in other studies dealing with the evaluation of RT-LAMP assays for the rapid diagnosis of COVID-19 .N gene was the most sensitive one followed by the ORF8 and lastly the ORF3a. Two samples with Cq values below 20 were not detected with the RT-LAMP for ORF8 and ORF3a, and one of these two was not detected by the N either. The sample which was negative for all the three genes by RT-LAMP was reanalyzed by RT-qPCR following the method described by the Institute Pasteur [E) along with the S gene designed in house (data not shown). In addition, due to shortage of RNA from the other misidentified sample, it was not possible to repeat the test ; however, the reported Cq value for the reference RT-qPCR internal control was 31.33 while this value tended to be in the range of 23–26 for the rest of the samples. However, LAMP analysis of the RNaseP gene did not show any significant effect of the RNA extract over the amplification; thus, this sample was finally classified as an ND (lack of amplification following the Institute Pasteur’s protocol is described above). In It was observed that the low sensitivity of the assay was due to 18 misidentified samples. These samples were positive by RT-qPCR but negative by RT-LAMP. A closer look at these data highlighted the fact that the average Cq of these samples was 36.83 ± 1.81, see Abbott ID Now COVID-19 test, the limit of detection in clinical samples is higher than that of RT-qPCR [Panbio COVID-19 Ag Rapid Test, the reported assay takes longer (30 vs. 15 min) but provides higher sensitivity as the antigen test only reaches values above 90% in samples with Cq values lower than 30, while the current assay achieves these values with Cq values lower than 35. Likewise, in a study from Nagura-Ikeda et al., molecular assays based on RNA detection were reported to be more sensitive than rapid antigen tests in saliva samples as they detected viral RNA in 50.5 to 81.6% of their samples applying different techniques (RT-qPCR and RT-LAMP), while only 11.7% were positive with the rapid antigen test they selected [Overall, the performance of the reported methodology is similar to other previously published rapid molecular tests. Similar to what happened with the  RT-qPCR . The spe RT-qPCR . The fac RT-qPCR . Similar RT-qPCR ,39,40. Aselected . It is wselected ; thus, tselected ,44,45.The selected three sets of LAMP primers demonstrated good performance for RT-LAMP detection of SARS-CoV2. The use of a fluorescent mastermix, which allowed us to perform result confirmation by melt curve analysis, further enhanced the specificity of the assay. The methodology was evaluated in a set of random nasopharyngeal clinical swab samples, indicating that they have an excellent specificity, and reaching sensitivity levels similar to those of other previously reported rapid nucleic acid-based assays alternative to RT-qPCR. Overall, the results indicate that the proposed method would be more suitable for frequent population testing in a constant surveillance mode, rather than as a clinical diagnostic tool.